Send Orders for Reprints to [email protected] 138 The Open Microbiology Journal, 2014, 8, 138-143 Open Access Acetate kinase Activity and Kinetic Properties of the Enzyme in Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 Intestinal Bacterial Strains Ivan V. Kushkevych* Laboratory of Molecular Biology and Clinical Biochemistry, Institute of Animal Biology of NAAS of Ukraine, Vasyl Stus St 38, Lviv 79034, Ukraine Abstract: Activity of acetate kinase in cell-free extracts and individual fractions and the kinetic properties of the enzyme obtained from the Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains were presented at the first time. The highest activity of the enzyme was measured in the cell-free extracts (1.52 0.163 and 0.46 0.044 U × mg-1 protein for D. piger Vib-7 and Desulfomicrobium sp. Rod-9, respectively) compared to other fractions. The specific activity of acetate kinase in the extracts of both bacterial strains was determined at different temperature and pH. Analysis of the kinetic properties of the purified acetate kinase was carried out. The acetate kinase activity, initial (instantaneous) reaction rate (V0) and maximum rate of the acetate kinase reaction (Vmax) in D. piger Vib-7 and Acetyl phosphate ADP Desulfomicrobium sp. Rod-9 intestinal bacterial strains were defined. Michaelis constants (Km and Km ) of the enzyme reaction (2.54 0.26 and 2.39 0.24 mM for D. piger Vib-7 as well as 2.68 0.25 and 2.47 0.27 mM for Desulfomicrobium sp. Rod-9, respectively) were calculated. The described results of acetate kinase, an important enzyme in the process of organic compounds oxidation and dissimilatory sulfate reduction would be perspective and useful for clarification of the etiological role of these bacteria in the development of inflammatory bowel diseases in humans and animals. Keywords: Acetate kinase, inflammatory bowel diseases, kinetic analysis, sulfate-reducing bacteria. INTRODUCTION Intestinal sulfate-reducing bacteria consume the organic compounds as a carbon source and electron donor in the process of dissimilatory sulfate reduction [1]. The species of Lactate oxidation to acetate occurs together with the Desulfovibrio and Desulfomicrobium genera oxidize these concurrent reduction of sulfate to sulfide [1, 2]. In the compounds incompletely to acetate via pyruvate and other intermediates [2]. The process of organic compounds presence of lactate and sulfate in the human intestine oxidation is a complex and multistage that provides the contributes to the intensive bacteria growth and the bacterial cells with energy. The lactate is the most common accumulation of their final metabolism products, acetate and substrate used by the species belonging to the intestinal hydrogen sulfide, which are toxic and mutagenic to epithelial sulfate-reducing bacteria [3]. intestinal cells [3, 8]. The increased number of the sulfate- reducing bacteria and intensity of dissimilatory sulfate In previous researches, it was demonstrated that the reduction in the gut can cause inflammatory bowel diseases lactate consumption by the intestinal sulfate-reducing of humans and animals [3, 8-10]. bacteria D. piger Vib-7 and Desulfomicrobium sp. Rod-9 and acetate accumulation in cultivation medium [4]. Lactate Acetate kinase from intestinal sulfate-reducing bacteria oxidation to acetate occurs with the intermediate compounds D. piger and Desulfomicrobium has never been well- formation: pyruvate, acetyl-CoA and acetyl phosphate [1, 2, characterized. In literature, there are some data on acetate 5]. kinase in various organisms as well as in the sulfate-reducing Acetate kinase (ATP:acetate phosphotransferase, EC bacteria isolated from environment [6, 11-18]. However, the 2.7.2.1) plays a significant role in energy production and data on the activity and the kinetic properties of this enzyme catalyzes the formation of acetate from acetyl phosphate. from intestinal sulfate-reducing bacteria Desulfovibrio piger This enzyme is involved in the synthesis of most of the ATP and Desulfomicrobium sp. has not been reported yet. formed catabolically [1, 6, 7]: The aim of this work was to study acetate kinase activity *Address correspondence to this author at Laboratory of Molecular Biology in cell-free extracts of intestinal sulfate-reducing bacteria D. and Clinical Biochemistry, Institute of Animal Biology of NAAS of piger Vib-7 and Desulfomicrobium sp. Rod-9 and to carry Ukraine, Vasyl Stus St 38, Lviv 79034, Ukraine; Tel: +380 32 270 25 04; out the kinetic analysis of enzymatic reaction. Fax: +380 32 270 23 89; E-mail: [email protected] 1874-2858/14 2014 Bentham Open Kinetic Properties of the Acetate Kinase in Intestinal Sulfate-Reducing Bacteria The Open Microbiology Journal, 2014, Volume 8 139 MATERIALS AND METHODS activity was expressed as U × mg-1 protein. The specific activity of the studied enzyme in the cell-free extracts of Objects of the study were sulfate-reducing bacteria both bacterial strains under the effect of different Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 temperature (+20, +25, +30, +35, +40, +45°C) and pH (5.0, isolated from the human large intestine and identified by the 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0) in the sequence analysis of the 16S rRNA gene [4, 19]. incubation medium was measured. Bacterial Growth and Cultivation Kinetic Analysis Bacteria were grown in a nutrition-modified Kravtsov- Kinetic analysis of the enzyme reaction was performed in Sorokin's liquid medium [4]. Before seeding bacteria in the a standard incubation medium (as it was described above) medium, 0.05 ml/l of sterile solution of Na2S×9H2O (1%) with modified physical and chemical characteristics of the was added. A sterile 10N solution of NaOH (0.9 ml/l) in the respective parameters (the incubation time, substrate medium was used to provide the final pH 7.2. The medium concentration, temperature and pH). The kinetic parameters was heated in boiling water for 30 min in order to obtain an characterizing the acetate kinase reaction are the initial oxygen-free medium, and then cooled to +30°C. The bacteria (instantaneous) reaction velocity (V ), maximum velocity of were grown for 72 hours at +37°C under anaerobic 0 the reaction (Vmax), maximum amount of the reaction conditions. The tubes were brim-filled with medium and product (P ) and characteristic reaction time (time half closed to provide anaerobic conditions. max saturation) were determined. The amount of the reaction product was calculated stoichiometrically. The kinetic Obtaining Cell-free Extracts parameters characterizing acetate kinase reactions such as Cells were harvested at the beginning of the stationary Michaelis constant (Km) and maximum reaction velocity of phase, centrifuged and suspended in 100 ml of 50 mM substrate decomposition were determined by Lineweaver- Tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH Burk plot [22]. For analysis of the substrate kinetic 7.0 (henceforth referred to as Tris buffer), containing 1 mM mechanism of acetate kinase, initial velocities were ethylenediaminetetraacetate (EDTA). A suspension of cells measured under standard assay conditions with different (150–200 mg/ml) was obtained and homogenized using the substrate concentrations. The resulting data were also ultrasonic disintegrator at 22 kHz for 5 minutes at 0 °C to analyzed by global curve fitting in SigmaPlot to model the obtain cell-free extracts. The homogenate was centrifuged kinetic data for rapid equilibrium rate equations describing for 20 min at 16,000 g to remove the cell debris. The pellet ordered sequential, V=(Vmax [A] [B])/(KA KB+KB [A]+[A] was then used as sedimentary fraction, and the supernatant [B]), and random sequential, V=(Vmax [A] [B])/(α KA KB+KB obtained was termed the soluble fraction. The supernatant [A]+KA [B]+[A] [B]), kinetic mechanisms, where V is the fluid and a Tris buffer wash of the pellet were subjected to a initial velocity, Vmax is the maximum velocity, KA and KB are second centrifugation at 16,000 g for 40 min [20]. The the Km values for substrates A and B, respectively, and α is soluble extract constituted by the supernatant was used as the the interaction factor if the binding of one substrate changes source of the enzyme. A pure supernatant, containing the the dissociation constant for the other [23]. soluble fraction, was then used as cell-free extract. Protein concentration in the cell-free extracts was determined by the Statistical Analysis Lowry method [21]. Kinetic and statistical calculations of the results were carried out using the software MS Office and Origin Assays for Acetate Kinase Activity computer programs. The research results were treated by the The acetate kinase activity was assayed colorimetrically methods of variation statistics using Student t-test. The as described previously in paper [17]. The reaction mixture equation of the straight line that the best approximates the of 1 ml contained 50 µmol imidazole buffer of pH 7.3, 800 experimental data was calculated by the method of least µmol potassium acetate, 10 µmol ATP, 20 µmol MgCl2, 200 squares. The absolute value of the correlation coefficient r µmol neutralized hydroxylamine and 0.5–1.0 U of acetate was from 0.90 to 0.98. The statistical significance of the kinase dissolved in 0.1 M phosphate buffer of pH 7.4 calculated parameters of line was tested by the Fisher’s containing 0.005 M cysteine. After incubation for l0 min at F-test. The accurate approximation was when P ≤ 0.05 [24]. 29°C the reaction was stopped by the addition of 1.0 ml 10% trichloroacetic acid. For the immobilization screening and RESULTS AND DISCUSSION storage stability tests, 1.0 ml of the same reaction mixture Specific activity of acetate kinase, an important enzyme was pipetted onto the enzyme-loaded glass beads.